Author:

Abstract:

In the Laboratory of Molecular Cell Biology, the sensing mechanisms resulting in the activation of the Ras-cAMP PKA pathway are studied both in Saccharomyces cerevisiae and Candida albicans. The S. cerevisiae G-protein coupled receptor Gpr1 together with the Gα protein Gpa2 are known to be responsible for activation of the cAMP pathway upon addition of glucose to yeast cells growing on a non-fermentable carbon source, and also seems to play a role in filamentous growth, together with the pheromone responsive MAP kinase cascade. We have recently shown that the S. cerevisiae G-protein coupled receptor Gpr1 directly interacts with glucose and sucrose (Lemaire et al., submitted). All experimental evidence suggest that Gpr1 acts on Gpa2 in a rather linear way. In 2002, it has been published that the C. albicans Gpa2 protein is most probably located upstream of the MAPK pathway (Sanchez-Martinez and Perez-Martin, Eukaryotic Cell). We have recently cloned and characterized the C. albicans Gpr1 homologue (see poster by Maydan et al.), and most of the obtained results indicate that Gpr1 acts upstream of the cAMP-PKA pathway. Using a two-hybrid assay we were then expecting to find an interaction between the CaGpr1 C-terminus and CaGpa2, as is the case in S. cerevisiae. Surprisingly, both proteins seem not to interact in C. albicans. For further investigation we then constructed a strain in which both GPR1 and GPA2 are deleted. The most important result is that under some conditions the phenotype of the double deletion strain is different from the phenotypes of the single deletion strains indicating that they act on parallel pathways. We performed adhesion assays on Caco2 cells and invasion assays on a 3-dimensional skin model. Virulence data, morphology experiments, adhesion and invasion data, and expression analysis of hyphal specific genes will also be presented. An important phenotype of the gpr1∆/gpr1∆ gpa2∆/gpa2∆ strain, is that this strain does not invade the agar when grown on solid SLAD medium, whereas the single gpr1∆/gpr1∆ and gpa2∆/gpa2∆ mutants do. We are now using this phenotype to screen a cDNA library for transformants that show restored invasive growth. This method should enable us to identify the downstream components of both CaGpr1 and CaGpa2.